Subject of the present invention is the preparation of polycation based bioconjugates that are suitable for transporting active substances of different type within the body, that is for functioning as carriers.
New polycation bioconjugates according to the invention are prepared by coupling [(k)Mx] and/or [(i)Mx] molecules, bearing functional groups appropriate for conjugationxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94to a given representative of isopolypeptide polycations, having free xcex1-amino groups, as carrier molecules, by chemical bonds; and the bioconjugates synthetized this way can be described by the general formula (I): 
and within the polycation bioconjugatis there are isopolypeptide polycation carrier molecules (further on: carrier molecules), having free xcex1-amino groups, that can be described by the general formula (I/a): 
and in each carrier molecule of general formula (I/a) there are monomeres of the same configuration (i.e. either D-, or L-), and the individual monomeres are not linked together by their amino groups in the xcex1-positions, but by their in other amino groups (i.e. in xcex2-, xcex3-, xcex4-, xcex5 etc.) positions, according to the value of xe2x80x9cmxe2x80x9d, and their structures are therefore divergent from those of the polypeptides build up by customary xcex1-amino-peptide bonds, generally occurring in mammal organisms;
wherein:
xe2x80x9crxe2x80x9d is a mean value between 20 and 400;
xe2x80x9cmxe2x80x9d=0, 1, 2, 3, . . . k;
xe2x80x9c[(k)Mx]xe2x80x9d designates enhancer molecules and/or connecting molecules conjugated by covalent (xe2x95x90k) bonds to the isopolypeptide polycation carrier molecule, and
xe2x80x9c[(i)Mx]xe2x80x9d designates enhancer molecules conjugated by ionic (xe2x95x90i) bonds to the isopolypeptide polycation carrier molecule, whereas the said enhancer molecules and connecting molecules having appropriate functional groups for conjugation may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kind and the enhancer molecules can be conjugated
directly and/or
indirectly through a connecting molecule,
and further the joint occurrence of [(k)Mx] and [(i)Mx] within the same polycation bioconjugate is symbolized by [(k/i)Mx]. On the basis of the general formula (I) of the new polycation bioconjugates according to the invention further molecules of general formulae (II), (III), (IV), (VI), (VII), (IX), (X), (XI) and of schematic formulae (V), (VIII), (IX/a), (X/a), (XI/a) can be derived.
In case the [Exi] enhancer moleculesxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are directly conjugated to a given representative of carrier molecules of general formula (I/a), by covalent bonds, then:
[(k)Mx]=[Exi]p1,
and the new polycation bioconjugates are being described by the general formula (II): 
In case the [(xe2x88x92)Cxj] connecting molecules of exclusively anionic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are conjugated to a given representative of carrier molecules of general formula (I/a), by covalent bonds, an additional possibility arises to establish ionic bonds with cationsxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94and then:
[(k)Mx]=[(xe2x88x92)Cxj]p2,
and the new conjugates are being described by the general formula (III): 
In case the [Exek] enhancer moleculesxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are indirectly conjugated by covalent bonds to a given representative of carrier molecules of general formula (I/a), through [Cxck] connecting moleculesxe2x80x94which may also be either identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94then:
[(k)Mx]=[Cxckxe2x88x92Exek]p3,
and the new polycation bioconjugates are being described by the general formula (IV): 
In the case when [Exi] and/or [Cxckxe2x88x92Exek] enhancer molecules and/or [(xe2x88x92)Cxj] connecting molecules of anionic character are also conjugated to a given representative of carrier molecules of general formula (I/a), and furthermore from among xe2x80x9cp1, p2 and p3xe2x80x9d the value of at least two are greater than 0, then:
[(k)Mx]=[Exi]p1+[Cxckxe2x88x92Exek]p3+[(xe2x88x92)Cxj]p2,
and the new polycation bioconjugates are being described by the schematic formula (V): 
wherein:
xe2x80x9cExxe2x80x9d in [Exi]p1 designates the Ex enhancer molecules of different (xe2x80x9cxxe2x80x9d) kind conjugated directly to a given representative of carrier molecules of general formula (I/a), by covalent bonds, and
xe2x80x9cixe2x80x9d indicates whether the Ex enhancer molecules, conjugated to the given carrier molecule by covalent bonds, are identical ones (i=1), or they are of different kind, of number xe2x80x9cixe2x80x9d (i=2, 3, . . . xe2x80x9cxxe2x80x9d kind); and
xe2x80x9c(xe2x88x92)Cxxe2x80x9d in [(xe2x88x92)Cxj]p2 designates (xe2x88x92)Cx connecting molecules of exclusively anionic character, of different (xe2x80x9cxxe2x80x9d) kind conjugated to a given representative of carrier molecules of general formula (I/a) by covalent bonds, in order to make it capable for establishing ionic bonds with cations, and
xe2x80x9cjxe2x80x9d indicates whether the (xe2x88x92)Cx connecting molecules, conjugated to the given carrier molecule by covalent bonds, are identical ones (j=1), or they are of different kind, of number xe2x80x9cjxe2x80x9d (j=2, 3, . . . xe2x80x9cxxe2x80x9d); and xe2x80x9cCxxe2x88x92Exxe2x80x9d in [Cxckxe2x88x92Exek]p3 designates the Ex enhancer molecules of different (xe2x80x9cxxe2x80x9d) kind, conjugated by covalent bonds indirectly, through Cx connecting molecules of different (xe2x80x9cxxe2x80x9d) kind, and these Cx molecules are also conjugated by covalent bonds to a given representative of carrier molecules of general formula (I/a), and
xe2x80x9cckxe2x80x9d indicates whether the Cx connecting molecules, conjugated to a given carrier molecule by covalent bonds, are identical ones (ck=1), or they are of different kind, of the number xe2x80x9cckxe2x80x9d (ck=2, 3, . . . xe2x80x9cxxe2x80x9d), and these Cx connecting moleculesxe2x80x94practically depending on the structure of the Ex enhancer moleculesxe2x80x94may be neutral and/or of anionic and/or of cationic character,
xe2x80x9cckxe2x80x9d indicates whether the Ex enhancer molecules, conjugated to a given carrier molecule indirectly through xe2x80x9cCxxe2x80x9d connecting molecules by covalent bonds, are identical ones (ek=1), or they are of different kind, of the number xe2x80x9cckxe2x80x9d (ek=2, 3, . . . xe2x80x9cxxe2x80x9d).
Furthermore the degree of saturation in % of a given representative of carrier moleculea of general formula (I/a) by [Exi]p1 and/or [Cxckxe2x88x92Exek]p3 enhancer molecules and/or [(xe2x88x92)Cxj]p2 connecting molecules are given by the different values of xe2x80x9cp1, p2 and p3xe2x80x9d, whereas the summarized value of xe2x80x9cp1+p2+p3xe2x80x9d within one given polycation bioconjugate is  greater than 0 and xe2x89xa6100; whereby the ratio between the free (not involved in peptide bonds) and bound NH2-groups is determined, which in turn influences the charge and the cationic character of the polycation bioconjugates; and thus
xe2x80x9cp1xe2x80x9d indicates a degree of saturation in % of a carrier molecule of general formula (I/a) with [Exi] enhancer molecules,
xe2x80x9cp2xe2x80x9d indicates a degree of saturation in % of a carrier molecule of general formula (I/a) with [(xe2x88x92)Cxj] connecting molecules of exclusively anionic character,
xe2x80x9cp3xe2x80x9d indicates a degree of saturation in % of a carrier molecule of general formula (I/a) with [Cxckxe2x88x92Exek] enhancer molecules which are bound to connecting molecules,
and on the basis of the above, in the schematic formula (V) xe2x80x9cp1+p2+p3xe2x80x9d greater than 0 and xe2x89xa6100, and from among xe2x80x9cp1, p2 and p3xe2x80x9d the value of at least two are greater than 0; further in a given polycation bioconjugate, the Ex molecules in [Exi] and the (xe2x88x92)Cx molecules in [(xe2x88x92)Cxj] are not necessarily identical with those Ex and Cx molecules occurring in [Cxckxe2x88x92Exek], which divergence is symbolized by xe2x80x9cxxe2x80x9d, and will be dealt with later at the examples of suitably selected enhancer molecules, further xe2x80x9crxe2x80x9d and xe2x80x9cmxe2x80x9d have the same meaning as in general formula (I).
In case the [(xe2x88x92)Axs] enhancer molecules of anionic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are directly conjugated by ionic bonds to the free xcex1-amino groups of a given representative of carrier molecules of general formula (I/a), then:
[(i)Mx]=[(xe2x88x92)Axs]t,
and the new polycation bioconjugates are being described by the general formula (VI): 
In case the [(+)Kxu] enhancer molecules of cationic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are conjugated directly through [(xe2x88x92)Cxj] connecting molecules of exclusively anionic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94by ionic bonds to a given representative of conjugates of general formula (III), then:
[(k)Mx]*[(i)Mx]=[(k/i)Mx]=[(xe2x88x92)Cxj]p2*[(+)Kxu]z,
and the new polycation bioconjugates are being described by the general formula (VII): 
In case additional [(xe2x88x92)Axs] enhancer molecules of anionic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are conjugated directly by ionic bonds to the free xcex1-amino groups of a given representative of polycation bioconjugates of general formula (VII), then:
[(k/i)Mx]={[(xe2x88x92)Cxj]p2*[(+)Kxu]z}*[(xe2x88x92)Axs]t,
and the new polycation bioconjugates are being described by the schematic formula (VIII): 
In case additional [(xe2x88x92)Axs] enhancer molecules of anionic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are conjugated by ionic bonds to the free xcex1-amino groups of a given representative of polycation bioconjugates of general formula (II) or (IV) or of schematic formula (V), then:
[(k/i)Mx]=[Exi]p1*[(xe2x88x92)Axs]t or [Cxckxe2x88x92Exek]p3*[(xe2x88x92)Axs]t or [Exi]p1+[Cxckxe2x88x92Exek]p3*[(xe2x88x92)Axs]t,
and the new polycation bioconjugates are being described by the general formula (IX), or by the schematic formula (IX/a): 
In case a given representative of polycation bioconjugates of schematic formula (V), in which there are [(xe2x88x92)Cxj] connecting molecules of anionic characterxe2x80x94which may also be either identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94and it thus gains partially anionic character, so that additional [(+)Kxu] enhancer molecules of cationic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94can be conjugated by ionic bonds to it, then: 
and the polycation bioconjugates are being described by the general formula (X), or by the schematic formula (X/a): 
In case additional [(xe2x88x92)Axs] enhancer molecules of anionic characterxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94are conjugated directly by ionic bonds to the free xcex1-amino groups of a given representative of polycation bioconjugates of general formula (X), then: 
and the polycation bioconjugates are being described by the general formula (XI), or by the schematic formula (XI/a): 
wherein:
xe2x80x9c(xe2x88x92)Axxe2x80x9d in [(xe2x88x92)Axs]t designates the (xe2x88x92)Ax enhancer molecules of anionic character, of different (xe2x80x9cxxe2x80x9d) kind conjugated directly to a given representative of carrier molecules of general formula (I/a), by ionic bonds, and
xe2x80x9csxe2x80x9d indicates whether the (xe2x88x92)Ax anionic/polyanionic molecules, conjugated to a given polycation carrier molecule by ionic bonds, are identical ones (sxe2x88x921), or, they are of different kind, of number xe2x80x9csxe2x80x9d (s=2, 3, . . . xe2x80x9cxxe2x80x9d kind), and
xe2x80x9c(+)Kxxe2x80x9d in [(+)Kxu]z designates (+)Kx enhancer molecules of different (xe2x80x9cxxe2x80x9d) kind of cationic character that are conjugated indirectly by ionic bonds, through [(xe2x88x92)Cxj] connecting molecules of different (xe2x80x9cxxe2x80x9d) kind of anionic character, to a given representative of carrier molecules of general formula (I/a), that is essentially to a conjugate of general formula (III), and
xe2x80x9cuxe2x80x9d indicates whether the (+)Kx cations and/or polyations, conjugated to a given compound of general formula (III) by ionic bonds, are identical ones (u=1), or they are of different kind of number xe2x80x9cuxe2x80x9d (u=2, 3, . . . xe2x80x9cxxe2x80x9d kind), and furthermore
xe2x80x9ctxe2x80x9d indicates a degree of saturation in % of a carrier molecule of general formula (I/a) with [(xe2x88x92)Axs] enhancer molecules, and
xe2x80x9czxe2x80x9d indicates a degree of saturation in % of a polycation bioconjugate general formula (I), or a carrier molecule of general formula (I/a) with [(+)Kxu] enhancer molecules, which are conjugated indirectly through [(xe2x88x92)Cxj] connecting molecules of anionic character, and
xe2x80x9ctxe2x80x9d in general formula (VI), and
xe2x80x9czxe2x80x9d in general formula (VII), and
xe2x80x9ctxe2x80x9d+xe2x80x9czxe2x80x9d in schematic formula (VIII), and
xe2x80x9ctxe2x80x9d+xe2x80x9cp1xe2x80x9d or xe2x80x9ctxe2x80x9d+xe2x80x9cp3xe2x80x9d or xe2x80x9ctxe2x80x9d+xe2x80x9cp1xe2x80x9d+xe2x80x9cp3xe2x80x9d in general formula (IX) and in schematic formula (IX/a), and
xe2x80x9czxe2x80x9d+xe2x80x9cp1xe2x80x9d or xe2x80x9czxe2x80x9d+xe2x80x9cp3xe2x80x9d or xe2x80x9czxe2x80x9d+xe2x80x9cp1xe2x80x9d+xe2x80x9cp3xe2x80x9d in general formula (X) and in schematic formula (X/a), and
xe2x80x9ctxe2x80x9d+xe2x80x9czxe2x80x9d+xe2x80x9cp1xe2x80x9d or xe2x80x9ctxe2x80x9d+xe2x80x9czxe2x80x9d+xe2x80x9cp3xe2x80x9d or xe2x80x9ctxe2x80x9d+xe2x80x9czxe2x80x9d+xe2x80x9cp1xe2x80x9d+xe2x80x9cp3xe2x80x9d in general formula (XI) and in schematic formula (XI/a), and the value of each of these sums is between  greater than 0 and xe2x89xa6100; furthermore according to these the NH2-groups which are not saturated by xe2x80x9ctxe2x80x9d and/or xe2x80x9czxe2x80x9d and/or xe2x80x9cp3xe2x80x9d and/or xe2x80x9cp3xe2x80x9d will remain free, whereby the ratio between the free (not involved in chemical bonds) and bound NH2-groups is determined, which in turn influences the charge and the cationic character of the polycation bioconjugates, whereas xe2x80x9czxe2x80x9d indicates the degree of saturation of carrier molecules with [(+)Kxu] cationic enhancer molecules that are conjugated indirectly through [(xe2x88x92)Cxj] connecting molecules, therefore xe2x80x9czxe2x80x9d=xe2x80x9cp2xe2x80x9d; furthermore
xe2x80x9crxe2x80x9d and xe2x80x9cmxe2x80x9d and xe2x80x9c[(k)Mx]xe2x80x9d have the same meaning as in general formula (I),
xe2x80x9c[Exi]p1xe2x80x9d has the same meaning as in general formula (II),
xe2x80x9c[(xe2x88x92)Cxj]p2xe2x80x9d has the same meaning as in general formula (III),
xe2x80x9c[Cxckxe2x88x92Exek]p3xe2x80x9d has the same meaning as in general formula (IV).
Novelty of the polycation bioconjugates of general formula (I), subject of the present invention, consists in that there are isopolypeptides having free xcex1-amino groups, as polycation carrier molecules in them, the synthesis of which is being carried out by coupling the diamino-monocarbonic acid monomers that build up these isopolypeptides, not by their amino groups in xcex1-positions, but their amino groups in other (xcex2-, xcex3-, xcex3-, xcex5- . . . etc.) positions, and the method used for preparation of these carrier molecules has been disclosed in the patent specification HU 202553 B, with the priority of Oct. 10, 1987, titled: xe2x80x9cProcess for preparation of isopolypeptides from diamino-monocarbonic acids and of drugs containing them, and a plant protection agent containing polyisolysinexe2x80x9d, further in the paper of Szxc3x3kxc3xa1n et al.: xe2x80x9cStructure Determination and Synthesis of Lysine Isopeptides Influencing on Cell Proliferationxe2x80x9d (Biopolymers, J. Wiley and Sons, Inc. 42:305-318, 1997).
To a given representative of carrier molecules of general formula (I/a) within the new polycation bioconjugates of general formula (I), prepared according to the invention, practically any organic and/or inorganic molecule having functional groups appropriate for conjugation can be coupled as a suitably selected enhancer molecule, in accordance with the method shown by general formulae (II), (III), (IV), (VI), (VII), (IX), (X), (XI) and by schematic formulae (V), (VIII), (IX/a), (X/a), (XI/a). All these enhancer molecules can favourably be chosenxe2x80x94with a non-limiting mannerxe2x80x94from the group of compounds listed hereinbelow:
hormones and hormone antagonists of different kind (steroid, protein, peptide, etc.), and active fragments of peptide hormones, and derivatives thereof;
saturated and unsaturated fatty acids, cholesterols, phospholipides (phosphoglycerides, sphingomyelins, etc.), and derivatives thereof;
nucleic acids/antisense nucleotides;
monosaccharides, oligosaccharides, and polysaccharides, and derivatives thereof;
vitamines, and their derivatives;
known antitumor drugs and active substances, and derivatives thereof,
amino acids, oligopeptides, polypeptides, further glycoproteins and lipoproteins, their fragments, and derivatives thereof.
The new polycation bioconjugates of general formula (I) prepared according to the method described in the invention, contain carrier molecules of general formula (I/a), and a given representative of these carrier molecules is conjugated by chemical (covalent and/or ionic) bonds with enhancer molecules, which are suitably selected, according to the intended effect (for example antiproliferative, antimicrobial, gene delivery, improving of the quality of the diagnostic magnetic resonance imaging, etc.), and these enhancer molecules may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kind, and due to the applicability of multiple enhancer molecules in a given polycation bioconjugate of general formula (I), manifold direct and/or indirect effects can be obtained simultaneously. A few examples for the compounds that can favourably be applied to this purpose are listed hereinbelow.
Compounds comprising a part of the direct enhancer moleculesxe2x80x94with non-limiting character:
compounds having antiproliferative effects, for example: cytosiatics used in the clinical practice, furthermore cytokines, which influence the division and differentiation of the cells (for example different growth factors, as well as antibodies produced against the receptors of these factors, interferons, etc.), furthermore peptides/proteins which inhibit the formation of new blood-vessels around the tumor cells (angiostatins, endostatins), furthermore nucleic acids/antisense oligonucleotides exerting antiproliferative effects on the malignantly transformed cells;
compounds having antimicrobial effects, for example: antiviral, antibacterial, antimycotical, antiprotozooneal, etc. compounds, used in the clinical practice, furthermore nucleic acids/complexed antisense oligonucleotides, which inhibit the replication of the microbes;
nucleic acids isolated or synthetized for the purpose of gene transfer, which are suitable for treating genetic diseases (for example cystic fibrosis);
compounds improving the quality of the diagnostic magnetic resonance imaging, for example: paramagnetic metal ions and complexes containing metal ions of that kind, especially molecule complexes of gadolinium (Gd) ion (for example dimeglumine salt of Gd-diethylene-triamine-pentaacetic acid);
compounds having immunomodulant effects (for example interleukins, tumor necrosis factors, etc.) which control a given function of the immune system; and
numerous suitably selected compounds with other effects, not defined herein, that can be used with definite purposes, as enhancer molecules.
Compounds comprising a part of the indirect enhancer molecules, which develop or increase selectivityxe2x80x94with non-limiting character:
in relation to the antiproliferative effects, for example: monoclonal antibodies having specific affinities to a surface antigen of a given tumor cell, as well as antibodies or any compound having affinity to those kind of receptors (for example transferrin receptor or folate receptor among the vitamins, etc.) which are present in a greater ratio on the surface of the tumor cells than of the normal (not malignantly transformed) cells;
furthermore in the relation to the antiproliferative and any other effects, aimed at, any compound which has specific affinity to a certain receptor occurring exclusively on the surface of a given normal cell only (this receptor does not exsist as a result of a pathological process), namely for example the asialoglycoprotein cell surface receptors of the hepatic cells (to which specifically links the terminal galactose of the macromulecules), or any other compound coupling receptors, which are present in greater ratio on the surface of a given target cells;
in general compounds which may link to a given target cell (for example microbes or infected cells with microbes, etc.) to achieve indirect enhancer effect.
Compounds comprising a part of the direct and simultaneously indirect enhancer molecules, with non-limiting character:
in relation to the antiproliferative effects, for example: hormones, hormone antagonists and derivatives thereof, especially from among the polypeptide hormones the humane choriogonadotropine hormone, which having antiproliferative effects, furthermore antibodies produced against receptors of growth factors of different kind, which are present in greater ratio on the surface of a given tumor cell than on other cells, and simultaneously exert antiproliferative effects towards given malignantly transfomed cells, furthermore immmunotoxines, which are produced against a given tumor cell;
in relation to the antimicrobial effects, for example; neutralizing antibodies which are produced against a given microbes (for example viruses, bacteriums, funguses, etc.), furthermore immunotoxines, which are produced against a given microbe.
New polycation bioconjugates of general formula (I) prepared according to the invention contain carrier molecules of general formula (I/a), and a given representative of these carrier molecules is conjugated with enhancer moleculesxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94that are suitably selected according to the above mentioned examples, and the conjugation of these enhancer molecules are symbolized by [Exi]p1 and/or [ . . . xe2x88x92Exek]p3 which indicates that the molecules are coupled by covalent bonds, furthermore [(xe2x88x92)Axs]t having anionic character and/or [(+)Kxu]z having cationic character indicate the molecules that are coupled by ionic bonds.
New polycation bioconjugates of general formula (I) prepared according to the invention contain carrier molecules of general formula (I/a), and a given representative of these carrier molecules is conjugated with enhancer moleculesxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94that are suitably selected according to the above mentioned examples, and the enhancer molecules can be conjugated directly and/or indirectly through connecting molecules, and the latter can couple enhancer molecules covalently or ionically, symbolized by [Cxckxe2x88x92 . . . ]p3 of the covalent ones, and by [(xe2x88x92)Cxj]p2 of the ionic ones, respectively, and these connecting molecules may suitably be chosenxe2x80x94with non-limiting characterxe2x80x94from dicarbonic acids, tricarbonic acids, carbohydrates, or amino acids, or peptide chain elongators.
New polycation bioconjugates of general formula (I) prepared according to the invention contain carrier molecules of general formula (I/a), and a given representative of these carrier molecules is conjugated with enhancer moleculesxe2x80x94which may either be identical ones or of (two or more i.e. xe2x80x9cxxe2x80x9d) different kindxe2x80x94that are suitably selected according to the above mentioned examples, and the conjugation of the enhancer molecules by covalent and/or ionic chemical bonds takes place directly and/or indirectly, in a determined ratio, preferably to reach a saturation of 10 to 100%.
Preferred representatives of carrier molecules of general formula (I/a) within the new polycation bioconjugates of general formula (I), according to the invention include those 60-120 membered, non-racemic polyiso-L-lysines, i.e. poly(xcex5)-L-lysine-hydrogen-bromides which themselves possess certain antiproliferative and antiviral effects, as it is disclosed in the patent specification HU 202553 B, of the Hungarian priority of Oct. 21, 1987.
Subject of the invention is the recognition that each of the new polycation bioconjugates of general formula (I), prepared according to the method described in the present invention, contains carrier molecules of general formula (I/a), and these carrier molecules (which themselves possess certain antiproliferative effects) are conjugated by chemical bonds with compounds having antiproliferative effects (some compounds, suitably selected to this purpose are listed above among the direct enhancer molecules), and the bioconjugates so obtained, are being successfully applicable for the treatment of malignancies, developing in mammal organisms (further: tumors), in se, or combined with known tumor-inhibiting methods, accepted in the clinical practice.
The additional enhancer molecules, resulting in developing an appropriately chosen selectivity, that have particularly been disclosed hereinabove (among the indirect enhancer molecules), linked to the bioconjugates prepared according to the invention, are increasing the concentration of active substances in the tumors, whereby the unwanted side-effects can be diminished, and thus the effectiveness of the treatment may further be increased.
Conjugates similar to those new polycation bioconjugates of antiproliferative effect, according to the present invention, have already been prepared earlier. Papers have also been published about them, from among which we would refer to some as follows hereinbelow:
Bogdanov-A. A Jr., Martin-C., Bogdanova-A. V. et al.: An adduct of cis-diamine-dichloroplatinum(II) and poly(ethylene glycol)poly(L-lysine)-succinate: synthesis and cytotoxic properties; Bioconjug-Chem. January-February 1996; 7(1): 144-9.
Di-Stefano-G., Busi-C., Derenzini-M. et al.: Conjugation of 5-fluoro-2xe2x80x2-deoxyuridine with lactosaminated poly-l-lysine to reduce extrahepatic toxicity in the treatment of hepatocarcinomas; Ital-J-Gastroenteral-Hepatol. April 1998; 30(2): 173-7.
Paprocka-M., Boratynski-J., Dus-D. et al.: Conjugation of the monoclonal antibody 17-1A with the nitroacridine compound C921 with the poly-L-lysine as an intermediate agent; Arch-Immunol-Ther-Exp-Warsz. 1997: 45(4): 343-9.
Salazar-A. M., Levy-H. B., Ondra-S. et al.: Long-term treatment of malignant gliomas with intramuscularly administered polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose: an open pilot study; Neurosurgery. June 1996: 38(6): 1096-103: discussion 1103-4.
Another recognition contained in the present invention is the feature or the carrier molecules of general formula (I/a), in the new polycation bioconjugates of general formula (I), that as being polycations, they are appropriate for transporting of, as well as introducing to the target cells the suitably selected nucleic acids of polyanionic character, as enhancer molecules, bound to them by ionic bonds, i.e. for gene transfer, using the effect that by conjugating further enhancer molecules by covalent bondsxe2x80x94details of which see hereinabovexe2x80x94resulting in developing an appropriately chosen selectivity, the new polycation bioconjugates are being linked selectively to the target cells, or in essentially higher ratio to them than to cells of other type.
Conjugates similar to those new polycation bioconjugates, according to the present invention, capable of gene transfer, have already been prepared earlier. Papers have been published about them in the scientific literature, from among which we would refer to some publicationsxe2x80x94as examplesxe2x80x94hereinbelow:
Erbacher-P., Roche-A. C., Monsigny-M., Midoux-P.: The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes; Biochim. Biophys. Acta. Feb. 21, 1997; 1324(1): 27-36.
Ferkol-T., Perales-J. C., Mularo-F., Hanson-R. W.: Receptor-mediated gene transfer into macrophages; Proc-Natl-Acad-Sci-USA. Jan. 9, 1996; 93(1): 101-5.
Kollen-W., Erbacher-P., Midoux-P. et al.: Glycosylated polylysines. Nonviral vectors for gene transfer into cystic fibrosis airway epithelial cells; Chest. June 1997; 111(6 Suppl): 95S-96S.
Liang-W. W., Shi-X., Deshpande-D. et al.: Oligonucleotide targeting to alveolar macrophages by mannose receptor-mediated endocytosis; Biochim-Biophys-Acta. Mar. 13, 1996; 1279(2): 227-34.
Schneider-H., Huse-K., Birkenmeier-G. et al.: Gene transfer mediated by alpha2-macroglobulin; Nucleic-Acids-Res. Oct. 1, 1996; 24(19): 3873-4.
Schwarzenberger-P., Spence-S. E., Gooya-J. M. et al.: Targeted gene transfer to human hematopoietic progenitor cell lines through the c-kit receptor; Blood. Jan. 15, 1996; 87(2): 472-8.
Sosnowski-B. A, Gonzalez-A. M., Chandler-L. A. et al.: Targeting DNA to cells with basic fibroblast growth factor (FGF2); J-Biol-Chem. Dec. 27, 1996; 271(52): 33647-53.
Stewart-A. J., Pichon-C., Meunier-L. et al.: Enhanced biological activity of antisense oligonucleotides complexed with glycosylated poly-L-lysine; Mol-Pharmacol. December 1996; 50(6): 1487-94.
Furthermore the new polycation bioconjugates which are suitable for gene transfer in the case of further conjugation with compounds having antiproliferative effects (the suitably selected compounds, detailed among the direct enhancer molecules) are suitable for more effective treatment of the tumors. Scientific reprints have been also published about these, from among which we refer to some as follows hereinbelow:
Cristiano-R. J., Roth-J. A. :. Epidermal growth factor mediated DNA delivery into lung cancer cells via the epidemal growth factor receptor; Cancer-Gene-Ther. January-February 1996; 3(1): 4-10.
Foster-B. J., Kern-J. A.: HER2-targeted gene transfer; Hum-Gene-Ther. Apr. 10, 1997; 8(6): 719-27.
Ginobbi-P., Geiser-T. A., Ombres-D., Citro-G.: Folic acid-polylysine carrier improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (M14) cells; Anticancer-Res. January-February 1997; 17(1A): 29-35.
Nguyen-D. M., Wiehle-S. A., Roth-J. A., Cristiano-R. J.: Gene delivery into malignant cells in vivo by a conjugated adenovirus/DNA complex; Cancer-Gene-Ther. May-June 1997; 4(3): 183-90.
Schachtschabel-U., Pavlinkova-G., Lou-D., Kohler-H.: Antibody-mediated gene delivery for B-cell lymphoma in vitro; Cancer-Gene-Ther. November-December 1996; 3(6): 365-72.
Shimizu-N., Chen-J., Gamou-S., Takayanagi-A.: Immunogene approach toward cancer therapy using erythrocyte growth factor receptor-mediated gene delivery, Cancer-Gene-Ther. March-April 1996; 3(2): 113-20.
Watanabe-N., Sato-Y., Yamauchi-N., Niitsu-Y.: Gene delivery into human cancer cells via transferrin receptor; Nippon-Rinsho. March 1998; 56(3): 724-30.
Another recognition also belongs to the subject of the present invention, namely that each of the new polycation bioconjugates of general formula (I) prepared according to the method described in the invention, contains carrier molecules of general formula (I/a) and these carrier molecules (which possess certain antiviral effects themselves) are conjugated, by chemical bond, with suitably selected compounds having antiviral effects, as direct enhancer molecules, and due to this increase the antiviral effect of the new polycation bioconjugates.
New polycation bioconjugates which are produced in the way described in the paragraph before, conjugated furthermore, by chemical bond, with suitably selected compounds which develop or increase selectivity to the target cells which are infected by the virus (the suitably selected compounds detailed among the indirect enhancer molecules) are suitable to increase the relative concentration of the new antiviral character polycation bioconjugates in the cells which are infected by the virus and due to it the efficacy of the treatment will be increased and side-effects greatly diminished. Conjugates similar to those new polycation bioconjugates of antiviral effect, according to the present invention, have already been prepared earlier. Papers have also been published about them, from among which we would refer to some as follows hereinbelow:
Di-Stefano-G., Colonna-F. P., Bongini-A. et al.: Ribavirin conjugated with lactosaminated poly-L-lysine: selective delivery to the liver and increased antiviral activity in mice with viral hepatitis; Biochem-Pharmacol. Aug. 1, 1997; 54(3): 357-63.
Fiume-L., Di-Stefano-G., Busi-C. et al.: Liver targeting of antiviral nucleoside analogues through the asialoglycoprotein receptor; J-Viral-Hepat. 1997: 4(6): 363-70.
Fiume-L; Di-Stefano-G; Busi-C. et al.: Hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-L-lysine. Synthesis of the carrier and pharmacological properties of the conjugate; J-Hepatol. February 1997; 26(2): 253-9.
Nakazono-K., Ito-Y., Wu-C. H., Wu-G. Y.: Inhibition of hepatitis B virus replication by targeted pretreatment of complexed antisense DNA in vitro; Hepatology. June 1996: 23(6): 1297-303.
Another recognition also belongs to the subject of the present invention, namely that each of the new polycation bioconjugates of general formula (I) prepared according to the method described in the invention, contains carrier molecules of general formula (I/a) and these carrier molecules are conjugated, by chemical bond, with compounds having different kind of antimicrobial effects (the suitably selected compounds, detailed among the direct enhancer molecules), and due to this, the new polycation bioconjugates significantly increase the antimicrobial effects surpassing the antiviral character, which was described in the three previous paragraph.
New polycation bioconjugates which are produced in the way described in the paragraph before, conjugated furthermore, by chemical bond, with compounds which develop or increase selectivity to the target cells which are infected by different kind of microbas (the suitably selected compounds, detailed among the indirect enhancer molecules) are suitable to increase the relative concentration of the antimicrobial character polycation bioconjugates in the cells which are infected by the given microbas and due to this the efficacy of the treatment will be increased and side-effects greatly diminished.
Another recognition also belongs to the subject of the present invention, namely that each of the new polycation bioconjugates of general formula (I) prepared according to the method described in the invention, contains carrier molecules of general formula (I/a) and these carrier molecules are conjugated, by chemical bond, with macromolecular paramagnetic contrast agents for example the gadolinium molecule complexes, and derivatives thereof, (the suitably selected compounds, detailed among the direct enhancer molecules), and due to this, the new polycation bioconjugates significantly improve the quality of the diagnostic magnetic resonance imaging, by increasing the contrast between the different kind of organs, tissues, as well as the different pathological alterations, for example tumors.
New polycation bioconjugates which are produced in the way described in the paragraph before, conjugated furthermore, by chemical bond, with compounds which develop or increase selectivity to the different kind of target organs or different pathological alterations (the suitably selected compounds, detailed among the indirect enhancer molecules), are suitable to increase significantly the relative concentration of the paramagnetic contrast character polycation bioconjugates in the different kind of organs, tissue, as well as, the different pathological alterations, and due to this further improve the quality of the magnetic resonace imaging.
Conjugates similar to those new polycation bioconjugates of paramagnetic character, according to the present invention, have already been prepared earlier. Papers have also been published about them, from among which we would refer to some as follows hereinbelow:
Opsahl-L. R, Uzgiris-E. E., Vera-D. R.: Tumor imaging with a macromolecular paramagnetic contrast agent:gadopentetate dimeglumine-polylysine; Acad-Radiol. September 1995; 2(9): 762-7.
Su-M. Y., Muhler-A., Lao-X., Naleioglu-O.: Tumor characterization with dynamic contrast-enhanced MR1 using MR contrast agents of various molecular weights; Magn-Reson-Med. February 1998; 39(2): 259-69.
Vera-D. R., Buonocore-M. H., Wisner-E. R. et al.: A molecular receptor-binding contrast agent for magnetic resonance imaging of the liver, Acad-Radiol. June 1995; 2(6): 497-506.
Vogl-T. J., Hoffmann-Y., Juergens-M. et al.: Experimentelle Evaluierung der kontrastmittelverstxc3xa4rken, hochauflxc3x6senden MR-Angiographie am Tiermodell. Gd-DTPA gegenxc3xcber Gd-DTPA-Polylysin; Radiologie. March 1996; 36(3): 254-62.
Another recognition also belongs to the subject of the present invention, namely that each of the new polycation bioconjugates of general formula (I) prepared according to the method described in the invention, contains carrier molecules of general formula (I/a) and these carrier molecules as polycations make suitable the polycation bioconjugates to get into the mammal organism, via transdermal transport by iontophoresis.
The new polycation bioconjugates which are trasported through the skin, exert their effects mainly in the different strata of the skin, and in the subcutan tissues, at the area of the iontophoresis, and certain amount of them act systemic. These kind of actions depend on the molecular size, the physico-chemical character and the type of the suitably selected direct and/or indirect enhancer molecule of the polycation bioconjugates, as well as, on the nature of the applied electric field.
The concentration of the new polycation bioconjugates, which are prepared according to the therapeutical aims (for example antiproliferative or antiviral effects, etc.) and which contain the suitably selected and above detailed direct and/or indirect enhancer molecules, increases at the place of the transdermal application, and due to this the local efficacy of the treatment will be greatly increased and side-effects diminished. If the aim is to achieve a systemic effect via transdermal application, the advantage will manifest in a constant, non invasiv administration of the polycation bioconjugates, wich avoid the gastro-intestinal system. Conjugates similar to those new polycation bioconjugates capable of transdermal application, according to the present invention, have already been prepared earlier. Papers have also been published about them, from among which we would refer to some as follows hereinbelow:
Turner-N. G., Ferry-L., Price-M. et al.: lontophoresis of poly-L-lysines: the role of molecular weight?; Pharm-Res. October 1997; 14(10): 1322-31.
Vanbever-R, Prausnitz-M. R., Preat-V.: Macromolecules as novel transdermal transport enhancers for skin electroporation; Pharm-Res. May 1997; 14(5): 638-44.
Another recognition also belongs to the subject of the present invention, namely that each of the new polycation bioconjugates of general formula (I) prepared according to the method described in the invention, contains carrier molecules of general formula (I/a), and these carrier molecules are conjugated with above detailed, direct and/or indirect enhancer molecules, which are suitably selected according to a given therapeutical purpose (for example antiproliferative or antiviral effects or gene therapy, etc.), and these new polycation bioconjugates are to be placed into cationic liposomes, and due to this the efficacy of the treatments will be increased and the side-effects greatly diminished. Conjugates similar to those new polycation bioconjugates which are suitable to be placed into cationic liposomes, according to the present invention, have already been prepared earlier. Papers have also been published about them, from among which we would refer to some as follows hereinbelow:
Gao-X., Huang-L: Potentiation of cationic Liposome-mediated gene delivery by polycations; Biochemistry. Jan. 23, 1996; 35(3): 1027-36.
Lee-R. J., Huang-L.: Folate-targeted, anionic liposome-entrapped polylysine-condensed DNA for tumor cell-specific gene transfer, J-Biol-Chem. Apr. 5, 1996; 271(14): 8481-7.
Mack-K. D., Walzem-R. L., Lehmann-Bruinsma-K. et al.: Polylysine enhances cationic liposome-mediated transfection of the hepatoblastoma cell line Hep G2; Biotechnol-Appl-Biochem. June 1996; 23 (Pt 3): 217-20.
Saldeen-J., Curiel-D. T., Eizirik-D. L. et al.: Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes; Diabetes. September 1996; 45(9): 1197-203.
Vitiello-L., Chonn-A., Wasserman-J. D. et al.: Condensation of plasmid DNA with polylysine improves liposome-mediated gene transfer into established and primary muscle cells; Gene-Ther. May 1996: 3(5): 396-404.
Zelphati-O., Szoka-F. C Jr.: Mechanism of oligonucleotide release from cationic liposomes; Proc-Natl-Acad-Sci-U-S-A. Oct. 15, 1996; 93(21): 11493-8
The biologically effective conjugates which have been described in the scientific reviewes of medicine, and cited above, contain carrier molecules, which are build up from diamino-monocarbonic acid monomers, namely lysines, that are coupled to each other by peptide bonds through amino groups in the xcex1-positions, therefore as a result of their synthesis, poly-(xcex1)-L-lysine is formed, and these facts support anyway the recognitions of the present invention, indirectly.
On the basis of all above aspects, the novelty of the invention is comprising in that each of the polycation bioconjugates of general formula (I), prepared according to the invention, contains carrier molecules of general formula (I/a), and these carrier molecules are build up from diamino-monocarbonic acid monomers, which are coupled by peptide bonds formed via the amino groups in the (xcex2-, xcex3-, xcex4-, xcex5- . . . , etc.) positions, corresponding to the value of xe2x80x9cmxe2x80x9d, and not through amino groups in the xcex1-position, and therefore, as a result of the synthesis xcex2-, xcex3-, xcex4-, xcex5- . . . , etc. polypeptides are forming, which are structurally entirely different from those polypeptides that have been described in the cited scientific reviews. The biological behaviour of the new polycation bioconjugates of general formula (I) will therefore be altered. For instance, they are more resistant against proteolytic enzymes, further the carrier molecules of general formula (I/a) themselves possess certain antiproliferative, antiviral activity, and as a consequence, the biological, effectiveness of the new polycation bioconjugates of general formula (I) is being modified favourably. The new polycation bioconjugates of general formula (I) according to the invention are being formulated as pharmaceutical preparates that are applicable perorally, parenterally, or transdermally, for systemic or topical use.